Project Summary/Abstract Staphylococcus aureus is an important bacterial pathogen that provokes a diverse range of human diseases, ranging from mild skin lesions to invasive and life-threatening infections. The virulence traits that characterize S. aureus include surface adhesins and glycopolymers, as well as secreted proteins, such as cytolysins, superantigens, and proteases, many of which play vital roles in immune evasion. S. aureus also produces nano- sized, spherical, bilayered, extracellular membrane vesicles (EVs) with distinct biologic activities. The production of EVs represents a secretory pathway common to mammalian cells, fungi, and bacteria that allows for cell-free intercellular communication, but the mechanisms underlying EV biogenesis in Gram-positive bacteria are poorly understood. S. aureus EVs encapsulate cargo that includes surface adhesins, lipoproteins, capsular polysaccharides, and exoproteins, including proteases and pore-forming toxins, which have been shown to play roles in bacterial virulence and the transmission of biologic signals to host cells. The overall goal of this project is to gain a better understanding of the biological role that S. aureus EVs play in the pathogenesis of staphylococcal infections. The project hypothesis is that EVs modulate bacterial pathogenesis by serving as a novel secretory pathway for S. aureus to transport toxins, cytoplasmic proteins, and lipoproteins and deliver them into host cells, while protecting the cargo from detection or destruction by the external environment. Preliminary data indicate that S. aureus EVs package an array of virulence factors, are taken up by macrophages, and are cytotoxic for a variety of host cells. Moreover, S. aureus EVs induce cleavage of caspase-1 and release of mature IL-1? and IL-18 in human macrophages in a lipoprotein-dependent manner. These results highlight the role of EVs in inflammasome activation, an important intracellular immune pathway that has been shown to play a crucial role in determining the outcome of S. aureus infections. Specific mechanisms whereby EVs are generated and the downstream effects of EV release from the bacterial cell are goals of this proposal. The specific aims of this application are to: (1) elucidate the mechanisms underlying EV biogenesis in S. aureus and the role of EVs in the SA cellular release of cytoplasmic proteins, lipoproteins, and capsule; (2) determine whether EVs act as a transport mechanism to deliver S. aureus virulence factors into host cells; and (3) investigate EV-induced inflammasome activation and its biological role in S. aureus infections. To accomplish these goals, a multidisciplinary approach will be taken that combines genetics, molecular biology, cell biology, immunology, electron microscopy, biochemistry, and animal infection models. These efforts are essential for gaining a functional appreciation of the role that S. aureus EVs play in the host-pathogen interaction. The knowledge obtained from this study will deepen our currently limited understanding of microbial EVs and have broad implications for our understanding of the pathogenesis of Gram-positive pathogens.